doi.org/10.26434/chemrxiv.10259552.v1 Probing the Complex Loading Dependent Structural Changes in Ultra-High Drug Loaded Polymer Micelles by Small-Angle Neutron Scattering Benedikt Sochor, Özgür Düdückcü, Michael M Lübtow, Bernhard Schummer, Sebastian Jaksch, Robert Luxenhofer Submitted date: 06/11/2019 • Posted date: 13/11/2019 Licence: CC BY 4.0 Citation information: Sochor, Benedikt; Düdückcü, Özgür; Lübtow, Michael M; Schummer, Bernhard; Jaksch, Sebastian; Luxenhofer, Robert (2019): Probing the Complex Loading Dependent Structural Changes in Ultra-High Drug Loaded Polymer Micelles by Small-Angle Neutron Scattering. ChemRxiv. Preprint. https://doi.org/10.26434/chemrxiv.10259552.v1 Drug loaded polymer micelles or nanoparticles are being continuously explored in the fields of drug delivery and nanomedicine. Commonly, a simple core-shell structure is assumed, in which the core incorporates the drug and the corona provides steric shielding, colloidal stability, and prevents protein adsorption. Recently, the interactions of the dissolved drug with the micellar corona have received increasing attention. Here, using small-angle neutron scattering, we provide an in-depth study of the differences in polymer micelle morphology of a small selection of structurally closely related polymer micelles at different loadings with the model compound curcumin. This work supports a previous study using solid state nuclear magnetic resonance spectroscopy and we confirm that the drug resides predominantly in the core of the micelle at low drug loading. As the drug loading increases, neutron scattering data suggests that an inner shell is formed, which we interpret as the corona also starting to incorporate the drug, whereas the outer shell mainly contains water and the polymer. The presented data clearly shows that a better understanding of the inner morphology and the impact of the hydrophilic block can be important parameters for improved drug loading in polymer micelles as well as provide insights into structure-property relationships. File list (2) download file view on ChemRxiv SANS CUR Formulations CSS model ChemRxiv-SI.pdf (2.74 MiB) download file view on ChemRxiv SANS CUR Formulations CSS model ChemRxiv.pdf (1.80 MiB)
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doi.org/10.26434/chemrxiv.10259552.v1
Probing the Complex Loading Dependent Structural Changes inUltra-High Drug Loaded Polymer Micelles by Small-Angle NeutronScatteringBenedikt Sochor, Özgür Düdückcü, Michael M Lübtow, Bernhard Schummer, Sebastian Jaksch, RobertLuxenhofer
Submitted date: 06/11/2019 • Posted date: 13/11/2019Licence: CC BY 4.0Citation information: Sochor, Benedikt; Düdückcü, Özgür; Lübtow, Michael M; Schummer, Bernhard; Jaksch,Sebastian; Luxenhofer, Robert (2019): Probing the Complex Loading Dependent Structural Changes inUltra-High Drug Loaded Polymer Micelles by Small-Angle Neutron Scattering. ChemRxiv. Preprint.https://doi.org/10.26434/chemrxiv.10259552.v1
Drug loaded polymer micelles or nanoparticles are being continuously explored in the fields of drug deliveryand nanomedicine. Commonly, a simple core-shell structure is assumed, in which the core incorporates thedrug and the corona provides steric shielding, colloidal stability, and prevents protein adsorption. Recently,the interactions of the dissolved drug with the micellar corona have received increasing attention. Here, usingsmall-angle neutron scattering, we provide an in-depth study of the differences in polymer micelle morphologyof a small selection of structurally closely related polymer micelles at different loadings with the modelcompound curcumin. This work supports a previous study using solid state nuclear magnetic resonancespectroscopy and we confirm that the drug resides predominantly in the core of the micelle at low drugloading. As the drug loading increases, neutron scattering data suggests that an inner shell is formed, whichwe interpret as the corona also starting to incorporate the drug, whereas the outer shell mainly contains waterand the polymer. The presented data clearly shows that a better understanding of the inner morphology andthe impact of the hydrophilic block can be important parameters for improved drug loading in polymer micellesas well as provide insights into structure-property relationships.
File list (2)
download fileview on ChemRxivSANS CUR Formulations CSS model ChemRxiv-SI.pdf (2.74 MiB)
download fileview on ChemRxivSANS CUR Formulations CSS model ChemRxiv.pdf (1.80 MiB)
FitParameter1Inthefollowing,thenameandtheobtainedfitparametersofeachusedIrenamodelwillbe2given. To take a possible polydispersity of themicelles into account, the core radiuswas3assumed to follow a gaussian distribution function. The presence of such a distribution4functionwillsmearoutthefeaturesathigherQ-values.5Sincetheexperimentallyobtainedcurvesarealreadyverysmearedandalmostfeatureless,6thefittedstandarddeviationsareabnormallyhigh.Thissmearingisduetoaconvolutionof7therealdistributionfunctionandasystematicalerrorofeveryneutronscatteringinstrument.8There are different routines and algorithms in literature,which can take the instrumental9smearing into account during fitting [Pedersen]. Unfortunately, the used data analysis10softwaredidn’tprovidethisoption.Hence,theobtainedstandarddeviationsofthegaussian11distributionaresystematicallyhigherandcannotbeusedforpolydispersityestimation.1213A-pPrOzi-A/CUR:10/014
2FunctionalPolymerMaterials,ChairforAdvancedMaterialsSynthesis,DepartmentofChemistryandPharmacyandBavarianPolymerInstitute,UniversityofWürzburg,Röntgenring11,97070Würzburg,Germany3FraunhoferInstituteforIntegratedCircuits,X-RayDevelopmentCenterEZRT,Flugplatzstraße75,90768Fürth,Germany4ForschungszentrumJülichGmbH,JülichCenterforNeutronScience(JCNS)atHeinzMaier-LeibnitzZentrum,Lichtenberstraße1,85747Garching,Germany5SoftMatterChemistry,DepartmentofChemistry,HelsinkiUniversity,00014Helsinki,Finland✢authorscontributedequally*correspondenceto:[email protected]:nanomedicine,nanoformulations,drugsolubilization,drug-polymerinteraction,drug-coronainterac-tionsABSTRACT:Drugloadedpolymermicellesornanoparticlesarebe-ingcontinuouslyexploredinthefieldsofdrugdeliveryandnano-medicine.Commonly,asimplecore-shellstructureisassumed,inwhichthecoreincorporatesthedrugandthecoronaprovidesste-ricshielding,colloidalstability,andpreventsproteinadsorption.Recently,theinteractionsofthedissolveddrugwiththemicellarcoronahavereceivedincreasingattention.Here,usingsmall-angleneutron scattering, we provide an in-depth study of the differ-encesinpolymermicellemorphologyofasmallselectionofstruc-turallycloselyrelatedpolymermicellesatdifferentloadingswiththemodel compound curcumin. This work supports a previousstudyusingsolidstatenuclearmagneticresonancespectroscopyandweconfirmthatthedrugresidespredominantlyinthecoreofthemicelleatlowdrugloading.Asthedrugloadingincreases,neu-tronscatteringdatasuggeststhataninnershellisformed,whichweinterpretasthecoronaalsostartingtoincorporatethedrug,whereastheoutershellmainlycontainswaterandthepolymer.Thepresenteddataclearlyshowsthatabetterunderstandingoftheinnermorphologyandtheimpactofthehydrophilicblockcanbe importantparameters for improveddrug loading inpolymermicellesaswellasprovide insights into structure-property rela-tionships.
solubleinwater.Therefore,aplethoraofmethodsisusedtoin-crease their solubility.[3] Polymermicelles are nanoscopic struc-turesformedbyamphiphilic(block)copolymers.[4]Inasimplifiedpicture,hydrophobicAPIsaredissolvedinthehydrophobiccore,whereasthehydrophilicshellactsasaprotectivelayertopreventprematuredisintegrationorunwantedproteininteractionsandtoensureasufficientwatersolubility.However,theactualsituationmay bemore complex as recently shown for a variety of drugloadedmicelles,asthenatureofthehydrophobicblockcansignif-icantlyaffectthedrugloading.[5,6]Aparticularlystrongeffectwasreportedforthedifferentsolubilizationbehaviorsofstructurallyvery similar poly(2-oxazoline) (POx) and poly(2-oxazine) (POzi)baseddrugdeliveryvehicles(Scheme1).
Scheme1:A)Schematicsynthesisofthestructuralisomerspoly(2-oxazo-line)s (POx) and poly(2-oxazine)s (POzi) by living cationic ring openingpolymerization(LCROP)of2-substituted2-oxazolinesand2-substituted2-oxazines;B)Schematicrepresentationoftheamphiphilictriblockcopoly-mersallbearingtwohydrophilicpoly(2-methyl-2-oxazoline)blocks(blue)anddifferenthydrophobiccores(yellow)aswellastheirmaximumloadingcapacity(LC)forcurcumin(CUR).
Small structural changes in the polymer sidechain and polymerbackboneofthehydrophobiccorecausedpronounceddifferencesin the solubilization capacity for different hydrophobic com-poundssuchascurcumin(CUR)[7-9]andpaclitaxel(PTX).[10-13]Theinvestigated formulationsareofparticular interestasextremelyhighCUR-loading>50wt.%wereobserved,whichishighlyunu-sualfordrug-loadedmicelles,sincetheygenerallysuffer,withno-tableexceptions[14,15],fromratherlowdrug-loadings<20wt.%.[16-18]Repeatedly,astrongerhydrophobiccontrastresultedinlowerdrug loadings in this family of amphiphilic block copolymers,clearlystressingthepoint that thesimplisticpictureofahydro-phobiccore,whichdissolveshydrophobicdrugs[6],mayoftenbeinadequate.[12,19-21]Sincethedrug loading (inwt.%vs.polymer)andfinaldrugsolubilization(ing/L)arecriticalparametersdictat-ing,toacertainextent,theclinicalpotentialofaformulation,acloser look at the interactions between polymeric drug carriersandsolubilizeddrughasrecentlyreceivedattention.[21-23]Thisin-cludesacriticalevaluationofthetraditionalcore-shellconceptasevidentbythedrug-inducedmorphologyswitchofPOxbasedmi-celles fromworm-like, tosphericalandraspberry-likestructureswith increasingPTX-loading (0 –50wt.%)[24,25]. In contrast, theformationofaworm-likemorphologywasobservedathigh-load-ing(50wt.%)ofthesamedrug-carrierloadedwithetoposideandaplatinum(Pt)-basedprodrug.[26]ItwasrecentlyconfirmedbyCal-lariet.al.usingsolidstatenuclearmagneticresonance(NMR)andendocytosisstudies[22],thatthedifferentmorphologiescanhavedirectimpactonbiologicalproperties.[27]Micellesatlow-loadingofaPt-baseddrughadaratherloosestructure,whereasthehigh-loadedmicellesweremuchmorecondensedwithaggregatedPt-speciessurroundedbyadenselypackedhydrophiliccorona.Thecellularuptakeofthesemicelles,bearingfructosemoietiesinthehydrophiliccorona,dependedonfructose-specificcellularuptaketransporters.Accordingly,endocytosiswassignificantlyhigheratlowerloadingduetotheless-restrictedinteractionoftheflexiblepolymer chains. In contrast, the apparently too densely packedfructosemoietiesathigher loading reduced thecellularuptake.Using solid-stateNMR, itwas recently reported that thehydro-philiccoronaisalsosignificantlyinvolvedinthedrug/polymerin-teractionsinPOx/POzimicelles,inparticularathigherdrugload-ings,whichimpededdissolutionofthelyophilizedpolymermicellepowders,whichcanbeunderstoodasaunusualsolidamorphousdispersions.[28] Moreover, using fluorescence spectroscopy andlifetimemeasurements,significantdifferencesforthemolecularenvironment of the incorporated drug were found at very lowdrug loadingwhereno involvementof thehydrophiliccorona isexpected.[13] Inspiredby this,wesetout todetermine if the in-volvementofthehydrophiliccoronainthisformulationanddis-tinctpolymer-drugspecificitiesobservedforPOxandPOzibasedCURformulations[10]alsoresultindifferentmicellarmorphologiesorsizes.Theanalyticaltechniquesutilizedsofarwerenotabletoaddressthesequestions.
Togainextensiveinsightsintothemicellarstructure,small-angleneutronscattering(SANS)curvesofCURsolubilizedwiththreedif-ferentPOxandPOzibasedamphiphiles(Scheme1)wereobtainedatvariouspolymer/CURratios.Followingthis,notonlymorpho-logical transitions froma distinct core-shell to a core-shell-shellmodelwith increasing CUR-loading could be observed, but alsothecontentofpolymer,waterorCURinthedifferentlayerscouldbeestimated.
PreparationofCUR-loadedmicellesCURloadedpolymermicelleswerepreparedbythinfilmmethodasdescribedelsewhere.[29]Briefly,ethanolicpolymer(20g/L)andcurcumin(5.0g/L)stocksolutionsweremixedinthedesiredratio.After complete removal of the solvent at 55 °C under a mildstreamofargon,thefilmsweredriedinvacuo(≤0.2mbar)foratleast20min.Subsequently,preheated(37°C),ultrapureH2OwasaddedtoobtainthefinalpolymerandCURconcentrationsasmen-tionedinthemaintext.Toensurecompletesolubilization,theso-lutionswereshakenat55°Cfor15minat1250rpmwithaTher-momixercomfort(EppendorfAG,Hamburg,Germany).Non-solu-bilizedcurcumin,ifany,wasremovedbycentrifugationfor5minat9.000 rpmwithaMIKRO185 (Hettich, Tuttlingen,Germany).CURquantificationwasperformedbyUV-Visabsorptionofsam-plesdilutedinethanolusingaBioTekEonMicroplateSpectropho-tometer (Thermo Fisher Scientific, MA, USA) and a calibrationcurveobtainedwithknownamountsofCUR.[10]ForSANSmeas-urements, the freshly prepared aqueous formulations werefreeze-driedandredissolvedindeuteratedwater(D2O)rightbe-fore measurements. Note that the densimetric measurementswereperformedinH2O.
DensimetryThe densimetric measurements were performed using a DMA4100Mdensitymeter (AntonPaar,Graz,Austria). The sampleswerediluted/dissolvedusingultrapurewater(H2O)andtheden-sityofeachsamplewasmeasuredfrom5°Cto55°C.Fortheesti-mationofthescatteringlengthdensities(SLD)insolutionandtocalculate the volume fraction, densities obtained at 25°C wereused.
Small-angle-neutron-scattering(SANS)TheSANSexperimentswereperformedontheKWS-1beamline[30]atHeinzMaier-LeibnitzZentrum(Garching,Germany).Thesam-plesweremeasured in standard Hellma quartz cuvetteswith apath-lengthof1mmandkeptat25°Cthroughouttheexperiment.Forthemeasurements,aneutronwavelengthof7Åwasused.TocoverthedesiredQ-range,thesamplesweremeasuredatthreesampletodetectordistancesof19.6m,7.6mand1.6mfor1200s,600 s and300 s respectively. Calibration to absolute intensitieswasdoneusingpoly(methylmethacrylate)(PMMA)asasecond-ary standard. For data correction, merging and reduction (azi-muthalaveraging)thetoolkitQtiKWSbyJCNSwasused.Theshapemodel (core-shell-shell sphere) is commonly used and readilyavailable inmost software. Themodel-dependent data analysiswascarriedoutusingthemacroIRENAforIgorPro.[31]
3.RESULTSANDDISCUSSIONDensimetryToestimate thevolume fractionsandneutron scattering lengthdensities (SLD) of polymer-CUR formulations at 25°C (at whichSANSexperimentswereconducted),thedensitiesoftheformula-tionsweredeterminedatthistemperature.Forthecompletetem-peraturedependentdensitydata, the reader is referred tosup-portinginformation(FigureS1-S6).Asexpected,thesolutionden-sityincreasedwithincreasingdrugconcentration(atconstantpol-ymerconcentration,Table1).
AsthepolymerandCURconcentrationswere≤1wt.%,itwasas-sumedthattheexcessvolume(polymer&CUR)duringmixingofthe samples is negligible. The obtained densities of the poly-mer/CURformulationswereusedtocalculatetherespectivevol-umefractions,𝜙.HerethepolymerandCURconcentrationcanbetransformedfromweighttovolumepercentusing:
where𝜌isthemacroscopicdensity,𝑏 theelement-andisotope-specificneutronscatteringlength,𝑚^ theelementspecificmolec-ular weight and 𝑛^ the stochiometric composition of the com-pound.FortheestimationoftheCUR-SLDthedensitywastakenfromliterature[33](Table2).
ThescatteringcurveofpureA-pPrOzi-AinD2OwithoutanyaddedCUR(A-pPrOzi-A/CUR=10/0,Figure1,A)showsaflatcurvewhichcan be described by the Debye function, indicating a Gaussianchain-like behavior, supporting earlier results, which suggestedthatthispolymerdoesnotformmicellesbyitselfunderambientcondition at this concentration (10 g/L).[29] Upon CUR addition,polymermicellesform,asshownbythechangeintheplateauin-tensitiesatlowQ-values,andtheoverallappearancesofthescat-teringcurves,indicativeofdiscreteandcompactobjects.Thein-creasingplateau intensitycanbecausedby largerparticlesorahigher scattering contrast. A recent report by Lübtow et al.[23]
showed that the hydrodynamic radii of A-pPrOzi-A-CUR aggre-gatesinitiallydecreaseslightlyatlowCURcontent(10/0.9:hydro-dynamicdiameter(Dh)=26nm;10-4.8:Dh=20nm)andonlystarttoincreaseatρ(CUR)>5g/L(10/11.9:Dh=46nm)asdeterminedbydynamiclightscattering(DLS).SinceDLSmeasuresthehydro-dynamicradius,whichinvolvesawatercoronaaroundtheparti-cle,andSANSprobestheradiusofgyrationwithoutthiscorona,differences are expected. The increasing scattering intensitiesprobably indicateahigherscatteringcontrastdue to thehigherCUR amounts, which is solubilized in the polymer micelles. At50wt.% drug loading, i.e. same concentrations of polymer anddrug,thescatteringintensityincreasesbynearlyanorderofmag-nitudecomparedtothepolymeralone(Figure1,AandB).Theal-readymentionedDLSresultsaswellascryo-TEMimages[29]havealsoshownthepresenceoflargerandworm-likeparticles.TheselargerstructureswerealsoobservablebySANS,astheincreasingscattering intensities at the lowestmeasuredQ-values indicate(Figure1,D).Toinvestigatetheseparticlesinmoredetail,power-lawormodel-basedfittingtechniquescouldbeused.However,foraccurateresults,eithertheirshapeandsizeortheexactratiobe-tweenmicellesandlargerparticlesmustbeknown.Inthepresentstudy,weconcentrateonthemorphologicalstudyofthesphericalmicellesandhenceonlythecorrespondingQ-rangeforsinglemi-celles(0.007-0.3Å-1)wasconsideredforfurtherdataanalysis.
Figure1:MeasuredSANSdataforA)A-pPrOzi-A,B)A-pBuOzi-AandC)A-pBuOx-AandtheirCURnanoformulations.Theconcentrationofthepoly-merwaskeptconstantat10g/L,whiletheCURconcentrationwasvariedfrom0to10g/L.InthecaseofA-pBuOx-A,CURconcentrationsabove3g/Lalreadycausedprecipitation.Onlyonethirdofthedatapointsisshownto increasevisibility.Thesolid linesare fits to thedataobtainedby thecore-shell-shellmodel(allfitparameterscanbefoundinthesupportinginformation);D)toshowthepresenceoflargeraggregates,thewholedatarangeextendedtoverysmallQ-valuesisshownforA-pPrOzi-Aatc(CUR)=1g/L(bottomright).Thedataanalysishowever,wasdoneonthecroppedQ-rangeshownintheothergraphsneglectingthoseaggregates.
ChoiceoffittingmodelFortheanalysisoftheSANSdata,threedifferentsphericalformfactor models were considered: A simple sphere, a core-shellsphereandacore-shell-shellsphere.EachmodelisavailableintheIrenamodellingsuiteandwasusedtofitthedata.Theresultingc2-valuesofthebestobtainedfitswereusedasanindicatorforthemostsuitablemodelfordataevaluation.Afullexampleandexplanationofonesampledatasetandthefittingresultscanbefound in the supporting information (Figure S7, S8). Based ontheseresults,thecore-shell-shellformfactormodel[31]
𝐹 𝑄 = rstuvt
𝜌: − 𝜌x 𝐽: 𝑄𝑅: + rs|uv|
𝜌x − 𝜌r 𝐽: 𝑄𝑅x +rs}uv}
𝜌r − 𝜌~ 𝐽: 𝑄𝑅r (5)
waschosenandusedtofitalldataforcomparability.Here𝑉:to𝑉rarethevolumesofeachcompartment(core,first(inner)orse-cond(outer)shell),𝑅:to𝑅rtheirrespectiveradii,𝜌:to𝜌rtheSLDofeachcompartment,𝜌~theSLDofthesolventand𝐽:istheBesselfunctionof the firstkind.Aschematicoverviewof thismodel isgiveninScheme2.
Scheme 2: A graphic representation ofthe employed core-shell-shell spheremodelwithitsparametersasaredefinedinEq.5.
Inadditiontotheformfactor,astructurefactorforsampleswithCURconcentrationsabove1g/Lwasusedfordescribingtheinter-micelleinteractions.Inthepresentanalysis,thehardspherestruc-ture factor[35-37] was used. This factor assumes a sphericallyshaped interaction potential between the particles. Hence, thesphere´sdiameter,𝐷,andvolumefractionofthespheres,𝜙,arenot parameters of the micelles, but of the modeled spheresaroundthem,whichrepresenttheirinteractionpotential.ForthehighestCURconcentration(10g/L),astickyhardspherestructurefactor[35]wasused,becausestickymicellesandinter-micellarcon-tacts were observed for A-pPrOzi-A/CUR = 10/10 g/L by cryo-TEM[29].
FitResultsThepossibleparametersetofthechosenmodelisratherlargein-cludingeight(withoutstructurefactor)ormorefitvariables.Forreasonsofclarity,onlythemicellarstructuredefiningparameters(sizeparametersandSLDs)willbeshown(Figure2and3).More-over,toconstrainthefittingprocedure,itwasattemptedtomatchtheoverallparticlesizewiththeresultsfromDLSandcryo-TEM.Thefulllistofmodelparametersforeachsamplecanbefoundinthe supporting information. As mentioned, A-pBuOzi-A and A-pBuOx-AformmicelleswithouttheneedofaddedCURwithcriti-calmicelleconcentration(cmc)valuesof5mg/L(0.5µM)and8mg/L(1µM),respectively.OnlyA-pPrOzi-AneedsCURtoformmi-celles, i.e. showsCUR-inducedmicellization.[29]Hence, therearenovalues for thecoreandshell sizesaswellas their respectiveSLDsforpureA-pPrOzi-AinheavywaterwithoutanyCUR.Obvi-ously,themicellarsizesandstructuresdevelopdifferentlyinde-pendenceoftheCUR-contentforallthreepolymers(Figure2,3).Therefore,thedifferentformulationswillbeevaluatedseparatelyinthefollowing.Importanttonote,theformulationofA-pBuOx-Aatpolymer/CUR=10/5isalreadyabovethemaximumdrugload-ing,andprecipitationoccurs.Thisresultedinveryunstablefitsinourcurrentwork.Therefore,wewillnotdiscussthedataanalysisofthisformulationatthisloadinganyfurther.
Figure2:Graphicrepresentationofthemicellarsizeparameters:A)core-radius,B)shell1thickness,C)shell2thicknessandD)totalmicelleradius.Pleasenote,whensizeparametersofaparticularcompartmentapproachzero, one can consider the resultingmorphology again as core-shell in-steadofcore-shell-shell.
foraCUR-concentrationof3g/L,whichhasnearlythesamesizeasthefirstshell(Figure2,C).Thisshellalsoincreases slightly in sizewithincreasingCURfeed.
Figure3:Graphicrepresenta-tionofthefittedSLDsofA)themicellescore,B) its firstshellandC)secondshell.Addition-ally, theSLDsofheavywater(blue dashed line), CUR (reddash-and-dot line) and thepolymers (black dotted line)aremarked.
At maximum loading(10/10),anothernotablechangeoftheA-pBuOzi-Amicellesisob-servedwith the thicknessof shell 1doubling in size toapproxi-mately60Å. Ingeneral,wefoundthatthedataanalysisatCUR
Apartfromthesize,furtherinsightsintotheactualcompositionofthedifferentmicellarlayerscanbeobtainedfromthefittedSLDs(Figure3).PureA-pBuOzi-A(CUR=0g/L)formssphericalaggre-gateswithnocore-shelldifferentiation.ThefittedSLDisapprox.3.6x10-6Å-2andthereforealmostperfectlyinbetweentheSLDsofA-pBuOzi-A and D2O (Table 2), suggesting that the ratio of A-pBuOzi-A/D2Oisroughly1/1.Inotherwords,thesemicellesdonotexhibitacore-shellstructurebutareratherhomogenousincom-position,whichwetentativelyattributetoexcellenthydrationofthehydrophobic repeatunitsbyvirtueof thepolarand flexiblepoly(2-oxazine)backbone.ByaddingCUR(CUR=1g/L),SLDcorede-creasesslightly(Figure3,A),whichcouldbeanindicationofthedehydrationofthecoreinfavorofCURinclusion.TheSLDofthefirstshellisslightlyabovetheinitialvalueofthesphericalaggre-gates(roughly4x10-6Å-2)(Figure3,B),whichhintstowardsaA-pBuOzi-A/D2OmixturewithaslightlyhigherD2O-fraction.These-cond,verysmall,shellcontainsalmostonlyD2OasjudgedbytheSLD(Figure3,C).BeingverysmallandessentiallyD2O,thissecondshellisnegligible.WithincreasingCURconcentration,theSLDsofthecoreandbothshellsdecreaseindicatingfurtherdehydration.AfterreachingaCURconcentrationof3g/L,theSLDsofthesys-temremainalmostconstant.Thisindicatesapossiblystablecom-positionineverypartofthemicelle.AccordingtotheSLDs,itap-pearsasthoughCURismostlypresentinthecore.AtthehighestpossibleCURconcentrationof10g/L,themorphologicalsituationissomewhatsimilartothesituationwithoutCUR,asthecomposi-tionofallcomponentsappearstobequitesimilar,accordingtotheSLDs(Figure3).Accordingly,wecannotconsiderthemicellesanymoreascore-shell-shellstructurebutratheralargehomoge-noussphere.AsimilardistributionofCURintotheouter,hydro-philicshellofglycopolymerswaspreviouslyobservedbyStenzeland coworkers using SANS.[23] Increasing amount of CUR dehy-dratedthenanoparticleshell,whichcoincidedwellwithalowercellularuptakeoftherespectivenanoparticles.
A-pBuOx-AIncontrasttoA-pBuOzi-A,neatA-pBuOx-Amicelleshaveacore-shellstructureandaresignificantlylargerthantheA-pBuOzi-Aas-sembliesatthesameconcentrationwithatotalradiusofapprox-imately75-80Å(Figure2,D).However,theSLDsofthecoreandthisshellarealsoverysimilar(Figure3,AandB).Again,thesizeofthe second shell is negligible, resulting in an overall core-shellstructure(Figure2,C).ThisseemsinconsistentasBuOzishouldbemorehydrophobicthanBuOx,andthus,astrongercore-shellcon-trastwouldbeexpected.However,preliminarycomparisonof1H-NMRspectrainCDCl3andD2OindeedsuggestahighlyhydratedandthusmobileBuOzicore,butthiswillhavetobestudiedsepa-ratelyinmoredetail.
Incontrasttotheothertwopolymers,A-pBuOx-AmicellesseemtoshrinkinsizeinthepresenceofCUR.AtaCURconcentrationof1g/L,theoverallsizeofthemicellesreducestoapproximately60Å,eventhoughasecondshellbecomesapparent(Figure2,CandD).Thiscanbeexplainedbysplittingoftheinitialshellintoshell1andshell2.Therefore,thethicknessofthefirstshellisreducedbyhalfandalsothecoresizeisreduced(Figure2,AandB).Suchacompaction of the aggregate structure could be a hint towards
strongpolymer-CURinteractions.Interestingly,strongerdrug-pol-ymerinteractionswererecentlysuggestedinthesystemA-pBuOx-A/CURcomparedtoA-pPrOzi-A/CURbyfluorescenceup-conver-sionstudies.[13]AlthoughA-pPrOzi-AenablesextremelyhighCUR-loadingsupto54wt.%,incontrastto24wt.%ofA-pBuOx-A[10],atlowloading,themolecularmobilityofCURwithinA-pBuOx-Awaslower than in A-pPrOzi-A. This was interpreted with stronger,moredefinedA-pBuOx-A/CURinteractions,whereasCURseemedtobemorelooselyincorporatedintoA-pPrOzi-A.
WithincreasingCURloading(10/3),thecoresizeofA-pBuOx-Are-mainsnearlyconstantwhile the firstandsecondshell thicknessslightlyincreasesforthenanoformulations.Thisis incontrasttothe other to polymer, where core slightly increases at thispoint.Thetotalmicelleradiusapproachesagain80Å(Figure2,D).Abovethisconcentration,CURstartstoprecipitate,whichresultsin the failureof the fittingmodel, sincemore thanoneparticlepopulationispresentinsolution.ConsideringtheSLDvalues,thecoreandfirstshellareheavilyandalmostequallywellhydratedintheabsenceofCUR(Figure3,AandB).Thesecondshellofnegli-giblesizeconsistsonlyofD2O(Figure3,C).Atlowloading(10/1),fittingrevealedthatthefirstandsecondshellexhibitsameSLD-values and therefore should have a similar composition. There-fore,asimplecore-shellmorphologycanbeassumed.This isanindicationofadehydrationofthecoreandthesecondshell.ThelowercoreSLDcanbeexplainedbythepresenceofCUR,whiletheSLDreductionofthesecondshellcouldresultfromahigherpoly-mercontent.This is ingoodagreementwiththeoverallsmallermicellarsize,andsignificantlyreducedcoresize(Figure2,AandD).FurtherincreasingtheCURconcentrationto3g/L,thecoreSLDreducestoapoint,whereitcanbeassumedthatthecoreisalmostentirelyconsistingofCURandA-pBuOx-AwithlittletonoD2Oleft(Figure3,A).TheSLDofthefirstshellreducesaswell,whiletheSLDofthesecondshell increases(Figure3,CandD).Thiscouldagain indicate an incorporation of CUR in the first shell and in-creasingD2Ofractionintheoutershell.
A-pPrOzi-AAtaCURconcentrationof1g/L,A-pPrOzi-Aexhibitsapronouncedcore-shell-shell structure with a relatively small core and outershell,butverybigfirstshell(Figure2).Thetotalmicelleradiusisroughly70-80Å(Figure2,D),whichisinreasonablygoodagree-mentwithdatafromDLS.[29]WithincreasingCURconcentrations,boththecoreandoutershellgrow,whiletheinnershellshrinks.ReachingaCURconcentrationof10g/L, thecoredimension in-creasesveryprofoundly,whichisinlinewithdatafromDLS[29].
TheSLDsofallA-pPrOzi-A-micellepartsdecreasewithincreasingCURconcentration(Figure3).Startingfromahighlyhydratedcoreandfirstshell,itisquicklyevidentthatthelargestamountofCURisstabilizedinthecoreofthemicelles,sincetheSLDsofthecoredecreasemuchsteeperandtheSLDstabilizesinbetweentheSLDsofpureCURandA-pPrOzi-A(Figure3,A).Theinvolvementofthefirst shell in the solubilizationofCUR is clearlyevidencedby itsSLD,whichissmallerthantheoneofthesecondshell(Figure3,BandC).TheSLDofthelargecorecorroboratesamixtureofCURandpolymer.Therelativelythinfirstshellremainshydratedasev-identbyalargerSLDvalue.ThesecondshellvanishesagainatthispointastheSLDisessentiallythatofpureD2O.
Figure4:CalculatedCURvol-umefractionsusingthefittedSLDsofthecoreandfirstshell(Figure3)forA)A-pPrOzi-A,B)A-pBuOzi-A and C) A-pBuOx-A. The total volume fractionwasconstrainedbythesumofboth 𝜙��v of the core andfirst shell being identical tothe reported loading capaci-ties[29]. Here, the volume ofeachmicellesection(coreandshell 1) was calculated usingthe structural parametersshowninFigure3.
Additionally,theresultsaresummarizedandsketchedinFigure5.WhilethecoremainlyreceivesCURatlow[CUR]=1-3g/L,thefirstshellmustincludesmallamountsofCURhereaswelltoobtainthemeasuredLC.WithincreasingCURfeed,bothcoreandshell1in-corporatemoreCUR.𝜙��vintheshellreachesestimatedvaluesofupto20-30%forallthreepolymers(3and5g/L).Onlyathighpolymerconcentrations,thevaluesfor𝜙��vbecomelessreason-able and trustworthy, since the nanoformulations either aggre-gatedandprecipitated(A-pBuOx-A)ortheparticleshapebecomesmoreheterogeneticduetothepresenceoflargeragglomeratesor
worm-likestructures(A-pPrOzi-AandA-pBuOzi-A).Nevertheless,theanalysisofSANSdataunambiguouslyshowsthatthemicellarshellisinvolvedinincorporatinglargeamountsofCURandplaysanessentialroleinthestabilizationprocess.Thiscorroboratesre-cent finding,where solid-stateNMR spectroscopyalso revealedinteractionofCURwiththeamidemoietiesinthehydrophilicco-ronaofA-pPrOzi-Awhichleadtoadecreaseindissolutionratesathigherloadings.[28]Inaddition,whenthehydrophilicblockswereexchanged to the slightly less hydrophilic poly(2-ethyl-2-oxazo-line),solubilizationcapacityofthecorrespondingABAtriblockco-polymersforCURandpaclitaxeldrasticallydecreased.[38]Similarly,stabilizationofCURandpaclitaxelusingamethacrylatebasedsys-temfeaturingfructosecontainingcoronaformingblockshasalsobeenpreviouslyreported.[22,23]
4.CONCLUSIONPoly(2-oxazoline)/poly(2-oxazine) based micelles have beenshowntobehighlyunusualastheyenableextraordinaryhighdrugloadingofmorethan50wt.% inselectcases. Increasingexperi-mentalevidencesuggeststhatthishighdrugloadingisintimatelylinkedwithinteractionsofthedrugwiththehydrophiliccorona.Here,weinvestigatedtheinfluenceoftheloadingofthreediffer-ent but structurally similar ABA triblock copolymers with themodel compound curcuminon themorphologyof the resultingmicelles.WhilewithoutCURnopronouncedcoreshellcharacterwasfound,additionofsmallamountsofCURenhancedthecon-trastbetweencoreandcorona.Inallcases,CURconcentratedinthecoreatlowdrugloadings.WithincreasingCURconcentrations,the picture becomesmore complicated and the scattering datacould not be reasonably fitted using the previously employedcore-shellmodel.Ourdatasuggestsacore-shell-shellmorphology,withpartsofthehydrophiliccoronafillingupwithCURandeffec-tivelyformingasecond,innershell,whiletheothershellremainshydrated and colloidally stabilizes themicelles.Withmore CURadded,thissituationeventuallybecomesunstable,finallycausingprecipitation.Thishappensalreadyatabout25wt.%drugloadingfor the A-pBuOx-A micelles, while those with poly(2-oxazine)basedBblockallowoveralldrugloadingof50wt.%.Theinsuffi-cientdifference inscattering lengthdensitybetweenthehydro-philic and hydrophobic block of the studied block copolymers
AcknowledgmentsFinancial support by the Deutsche Forschungsgemeinschaft isgratefullyacknowledged(Projectnumber398461692,awardedtoR.L.).Theauthors thankJCNSforallocatingbeamtime(proposal#12083)andprovidingexcellentequipmentandsupport,before,during and after the beam-time. Also, we appreciate valuablefeedbackbyAnn-ChristinPöppler.M.M.L.wouldliketothanktheEvonikFoundationforprovidingadoctoralfellowship.
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